The broad objective of this proposal is to gain understanding of the physical mechanisms of the reactions between proteins and nucleic acids. We will use as a model system the regulatory interactions of the lac operon of E. coli. This system presents the desirable features and offers the experimental approaches necessary to test directly some of the mechanisms that have been proposed. One of the crucial questions that we want to address is the mechanism by which sequence-specific proteins reach their target site on the genome in the midst of a vast excess of other DNA sequences. A particular situation that we will also examine is the possible role of sequences which present striking analogies with and are situated near the target site. The influence that the binding of one protein at its site might have on the interaction of another protein at a nearby site (telestability) will also be investigated. We propose experiments which should allow the distinction between the "linear diffusion" model and the "direct transfer" mechanism that have both been invoked to account for the very fast associated of lac repressor and operator. We will also examine the effect of deleting each of the two operator-like regions, located in the vicinity of the lac operator, on the kinetics of the Lac repressor-operator interaction. We will measure the equilibrium and kinetic constants of the interaction of the CAP protein with its site on the lac promoter, and the possible effect of this interaction on the stability of the repressor-operator complex. The methodology for all of the above experiments involves the filter bonding assay and the use of purified repressor, CAP protein and labelled restriction fragments. We will seek direct physico-chemical evidence for the existence of two DNA binding sites on the lac repressor. This will involve the detection of an increase in molecular weight of repressor-operator complex as the result of the binding of another DNA molecule to the repressor.